Thermal refugia and persistence of Texas horned lizards (Phrynosoma cornutum) in small towns

Abstract Vegetation loss is a primary cause of habitat degradation and results in a decline in reptile species abundance due to loss of refuge from predators and hot temperatures, and foraging opportunities. Texas horned lizards (Phrynosoma cornutum) have disappeared from many areas in Texas, especially from urbanized areas, probably in large part due to loss of suitable habitat. This species still occurs in some small towns in Texas that still contain suitable habitat. Long‐term data from Kenedy and Karnes City, Texas indicate that when study sites experienced significant shrub and vegetation removal horned lizards declined by 79%. We hypothesize the decline was due to the degradation of the thermal landscape for these lizards. We determined the preferred temperature range (T set25 – T set75) of lizards at our study sites and took field measurements of body temperature (T b ). Temperature loggers were also placed in three microhabitats across our study sites. Shrubs and vegetation provided the highest quality thermal environment, especially for about 5 h midday when temperatures in the open and buried under the surface in the open exceeded the lizards' critical maximum temperature (CTmax) or were above their preferred temperature range. Horned lizard density was positively related to the thermal quality of the habitat across our sites. Texas horned lizards in these towns require a heterogeneous mix of closely spaced microhabitats and especially thermal refugia, such as shrubs and vegetation along fence lines and in open fields. Maintaining thermal refugia is one of the most important and practical conservation actions that can be taken to help small ectotherms persist in modified human landscapes and cope with increasing temperatures due to climate change.


| INTRODUC TI ON
Vegetation loss is a primary cause of habitat degradation and declines in reptile species abundance (Attum & Eason, 2006;Fleischner, 1994;Smith et al., 1996). This loss results in reduced prey diversity and number, increased predation risk due to loss of cover, and a reduction in important microhabitats required for thermoregulation (Jones, 1981;Norbury, 2001). Loss of vegetation leads to reptiles having to travel further to find prey, making them more vulnerable to predation (Hinsley, 2000;MacArthur & Pianka, 1966).
Vegetation loss also reduces the availability of microhabitats for thermoregulation, hindering the ability of lizards to escape lethal substrate temperatures which is critical for species to persist in harsh and arid habitats (Adolph, 1990;Attum et al., 2013;Carrascal et al., 1992). When lizards are exposed to temperatures greater than their preferred body temperature, their activity is restricted, making them vulnerable to extinction due to climate change (Sinervo et al., 2010). The lower foraging efficiency caused by higher temperatures decreases the number and quality of offspring produced, leading to declining populations and eventual extinction (Sinervo et al., 2010).
Microhabitats that offer temperatures suitable for lizards determine how far they must move and how much energy they expend in finding an ideal thermal environment (Grbac & Bauwens, 2001;Sears et al., 2016). Reptile activity patterns are constrained by the distribution of microhabitats across space and time (Grbac & Bauwens, 2001). Heterogeneous landscapes support microhabitats with thermoregulatory patches that are variable in temperature and spatially closer together (Sears et al., 2016). This microhabitat configuration allows lizards to expend less energy moving to a favorable thermal patch to regulate body temperature and allows more time for foraging and reproductive opportunities (Sears et al., 2016).
Homogeneous landscapes decrease available microhabitats and increase the distance lizards must travel between sun and shade, increasing their exposure to predators. Therefore, an understanding of the thermal regimes in different microhabitats is important to understand thermoregulatory behavior, habitat quality, and cost of living in diverse types of environments.
Lizards living in urban areas face additional challenges with thermal environments. Urban areas are often warmer and warm faster than natural areas due to concrete surfaces lowering albedo rates, thus increasing surface temperatures (Ackley et al., 2015;Kolbe et al., 2016;Taha, 1997). Research has shown that different types of landscaping in urban areas can significantly affect whether temperatures are within preferred temperature ranges for lizards (Ackley et al., 2015). Landscaping style (e.g., types of vegetation planted, fencing and borders, extent of tree canopy cover) can result in maximum daily air temperature differences up to 10°C between two adjacent habitats (Robinson et al., 2013;Todd & Andrews, 2008) and reduce surface temperatures over 10°C during the day (Brazel et al., 2007). Landscaping can also create habitat that can increase the diversity and abundance of reptiles in human modified areas (Ackley et al., 2015;Nopper et al., 2017;Pulsford et al., 2017). Few studies, however, have evaluated the thermal quality of microhabitats reptiles use in urban areas or how this might be related to abundance.
These temperature tolerances are higher and more variable than other sympatric species of desert lizards (Pianka & Parker, 1975).
Due to their ecology of being an ant specialist, it is likely that relaxed thermoregulation allows horned lizards to withstand direct sunlight for longer periods of time while foraging for ants in the open. Their cryptic camouflage and ability to withstand higher temperatures for longer aids in reducing predation risk since they do not have to move as frequently between sun and shade (Guyer & Linder, 1985;Pianka & Parker, 1975).
The Texas horned lizard is a threatened species in the state of Texas due to widespread population declines and a virtual disappearance in the eastern part of the state (Donaldson et al., 1994). These declines are attributed to habitat loss due to urbanization and agriculture, introduction of the red imported fire ant (Solenopsis invicta), loss of their preferred prey harvester ants (Pogonomyrmex spp.), and over-collecting for the pet trade (Dixon, 2000;Donaldson et al., 1994;Henke, 2003). The species has remained a wildlife component of some small towns in Texas in areas that have suitable habitat, including Kenedy and Karnes City in south Texas that we have monitored since 2013. Lizards can occur at higher densities at some sites in town (52 lizards/hectare; Ackel, 2016) than are observed in more natural areas (3-10 lizards/hectare; Whitford & Bryant, 1979;Whiting et al., 1993). Our research has shown that the high density of lizards observed in these towns may be due to a variety of factors including isolation due to roads and buildings (which could increase horned lizard densities due to limited dispersal; Wall, 2014), a dietary shift to consuming smaller more abundant prey items (Alenius, 2018), and reduced predation pressure compared to natural areas by some types of predators (Mirkin et al., 2021).
We have noticed during our studies that horned lizards disappear or decrease in density at sites that have had shrubs and brush piles removed. The removal of shrubs could result in decline if it increases predation or degrades the thermal landscape (Gaudenti et al., 2021).
Because predation pressure is lower in these towns than in more natural areas (Mirkin et al., 2021), we hypothesized that the primary cause of these declines is due to the loss of thermal refugia. fences, and vegetation (i.e., tree canopy cover, ornamental shrubs, and native vegetation) and fields, which have short vegetative cover interspersed with clumps of trees and shrubs ( Figure 1).
One of the study's authors (

| Body temperature in the field (T b )
In 2019-2021, field cloacal temperature (T b ) was recorded within using Esri ArcGIS® Pro. Study sites are split up into two types of spatial structure: (c) alleyways, which are more heterogeneous in structure and thermal microhabitats, and (e) have dirt roads bordered by houses, fences, and vegetation (i.e., tree canopy cover, ornamental shrubs, and native vegetation), and (d) fields, which are less heterogenous and have thermal microhabitats spread apart since fields have (f) short vegetation cover interspersed with clumps of trees and shrubs.

| Operative environmental temperatures (T e ) and model calibration
In 2019-2021, we determined environmental temperatures (T e ) at sites that currently have horned lizards and sites where they have disappeared. T e has historically been determined using copper or polyvinylchloride (PVC) models to estimate available temperatures for small ectotherms, but we used 3D printed models of adult Texas horned lizards for morphological accuracy (Mirkin et al., 2021;Watson & Francis, 2015). Models were printed with acrylonitrile butadiene styrene (ABS) and painted with 33% reflective paint (Rustoleum™ gray primer) that corresponds to the reflectivity of horned lizards (Adolph, 1990;Lara-Reséndiz, Gadsden, et al., 2015).
The underside of the model had a recessed opening that held a DS1922L Thermochron™ temperature logger that records temperature at a resolution of ±0.2°C ( Figure 2). Self-fusing repair tape was used to seal the temperature logger in the model. The addition of this tape did not significantly change the temperatures recorded by the temperature loggers (models with and without tape, y = 0.97x, n = 29, R 2 = .999).
After we could purchase more temperature loggers, 30 models were placed at 10 sites from June 30 -July 6th and August 1 -8th. In 2020 and 2021, 45 models were placed at 13 sites from June 1-June 14th, July 1 -6th, and August 1 -6th. Of these 13 sites, 11 were sites that were regularly surveyed and two of the sites were fields in Kenedy where lizards historically occurred but disappeared in the late 1990s (Wade Phelps personal communication; Table 2). We checked those two sites several times every year since 2013 for horned lizards and scat but never found evidence they had recolonized those areas. At each site, one model was placed in the open, one under vegetation (shrubs at 12 sites and thick grass under tree canopies at 3 sites), and one buried ~2 cm under the soil surface in open areas to mimic the three common microhabitats Texas horned lizards utilize (Burrow et al., 2001;Wall, 2014). The two largest sites had two sets of three models (n = 6) placed as far apart as possible to provide better coverage (

| Preferred body temperature in the laboratory (T set )
In 2021, T set was recorded using a laboratory thermal gradient that consisted of a plastic box 88.6 cm × 42.2 cm × 15.6 cm (length, width, and height) that was filled with 2-3 cm of sand (Angilletta, 2009;Hertz et al., 1993;Sinervo et al., 2010). At one end of the box, a  (Hertz et al., 1993;Lara-Reséndiz, Gadsden, et al., 2015).

| Habitat thermal quality and thermoregulatory indices
Following methodology from Hertz et al. (1993), data from T b , T set , and T e were used to calculate the accuracy of thermoregulation Given that d e did not vary between years (One-way ANOVA, F 2,223 = 2.2, p = .11), we averaged d e across years to obtain d e . An E value near to one indicates an organism that actively thermoregulates because environmental temperature is far from its preferred temperature. These lizards are under thermal stress and must increase or decrease their T b with respect to T e . An E value equal or near to zero indicates a thermoconformer, which is not regulating temperatures actively since the environmental temperature is within its preferred temperature range (Hertz et al., 1993).
However, an E value can come from a variety of combinations of d b and d e (Hertz et al., 1993).

| Change in density
Horned lizard density averaged 28.67 ± 7.15 individuals at our sites (n = 16 sites, range 1.23-93.9; Table 2). Horned lizard density was higher in alleys (55.09 ± 8.80, n = 7) than in fields (8.11 ± 2.13, n = 9) (ttest unequal variances, t 7 = 5.19, p = .001). There were only four sites which did not experience major vegetation removal between 2013 and 2021, and horned lizards declined an average of −0.04 ± 0.08% at these four sites. Twelve sites experienced major vegetation and brush removal between 2013 and 2021 ( Table 2). At four of these sites, vegetation that was removed grew back, and lizards recolonized one of these sites. At eight sites, the vegetation removed did not grow back and all lizards disappeared at five of those sites. The density of lizards at a site decreased by 79% in the year after vegetation removal (median density before removal = 22.75; median density after removal = 2.08, W = 78, n = 12, p = .002; Table 2).

| Body temperature in the field (T b )
One hundred and fifty-three P. cornutum were captured (

| Preferred body temperature in the laboratory (T set )
Individual as a random variable did not explain variability in T set temperatures (p = .07). Time of day nested in month (F 15,80 = 0.98, p = .49) and month (F 2,16 = 1.99, p = .17) also had no significant effect on T set .
Model predicted means averaged to 36 ± 0.47°C. We decided to use the observed mean of 35.7 ± 0.33°C since it was not statistically different than the model predicted mean (t 0.05(2),26 = 0.45, p = .65).

| Operative environmental temperatures (T e )
There was a highly significant linear relationship between field T b and model estimated T e (y = 0.80x + 6.57, R 2 = .89, p = .02) and the slope was not significantly different than 1.0 (n = 71, t 0.05 (2)  above preferred temperatures or sometimes above CT max (Figure 4).
Vegetation temperatures never reached T set in 2021 and stayed below their preferred temperature range the entire day ( Figure 4).
Ambient air temperatures were closest to temperatures found under vegetation, which is expected since temperature data are measured in the shade (Figure 4).
There was a significant difference in percent time that T e was at critical maximum temperature (F 2,323 = 13.09, p < .0001) and percent time that T e was at preferred temperatures (F 2,323 = 2.95, p = .05) between years. This difference was due to 2021 being on average cooler than 2019 and 2020 (Tukey, p < .05 both cases). After looking at temperature abnormalities at our field sites, 2021 was the only year that temperatures were cooler on average since monitoring this population starting in 2013 (NOAA Climate at a Glance: Global Time Series). We therefore decided to remove 2021 T e from the percent time at critical temperature and percent time at preferred temperature analyses below to give a more representative view of the temperatures commonly experienced by lizards at our sites.
Buried in the open microhabitat was above their critical temperature (CT max ) for 15.6% of the day and within their preferred temperature range (T set25 -T set75 ) for 20.7% of the day. Open microhabitat was above their critical temperature for 39.1% of the day and within their preferred temperature range 13.5% of the day.
Vegetation microhabitat was above their critical temperature for 0.3% of the day and within their preferred temperature range 25.1% of the day ( Figure 5). All microhabitats were significantly different from each other for percent time at critical temperature ( Figure 5;

| Thermoregulatory indices
The average deviations of T b from T set range (i.e., d b ) was low  Table 3). Effectiveness of thermoregulation (E) was 0.31, indicating P. cornutum is a moderate thermoregulator. Effectiveness of thermoregulation for d e − d b was 0.71, also indicating some thermoregulatory behavior and a thermally benign environment ( Table 3).

| DISCUSS ION
In tropical and desert areas the major challenge for lizards is to lower their body temperature and vegetation plays a key role in providing shade and cooling temperatures (Grimm-Seyfarth et al., 2017;Kearney et al., 2009). Urban areas located in these environments have additional challenges with increased surface temperatures, although landscaping can significantly affect whether temperatures are within preferred temperature ranges for lizards (Ackley et al., 2015). We found that shrubs in town pro-  (Alenius, 2018;Nutting et al., 1974;Scheffrahn & Rust, 1983). Ants are also sensitive to increased midday temperatures and have a bimodal pattern of activity (Whitford et al., 1980(Whitford et al., , 1981Whitford & Bryant, 1979 (Burrow et al., 2001;Whitford & Bryant, 1979). Burrowing is effective at making the lizards invisible, so it may more often function as a predator avoidance strategy when lizards are inactive. Texas horned lizards will also climb onto the trunk or lower branches of shrubs during the hottest times of the day for thermoregulation (Burrow et al., 2001;Whitford & Bryant, 1979). We have never observed this behavior in Kenedy or Karnes City, so we did not place models in TA B L E 3 Field body temperature (T b ), operative environmental temperature (T e ), preferred temperature in laboratory (T set ) and T set range (T set25 -T set75 ) in °C, and accuracy of thermoregulation (d b ), habitat thermal quality (d e ), and thermoregulatory effectiveness (d e − d b and E  Gaudenti et al., 2021;Grimm-Seyfarth et al., 2017;Ivey et al., 2020;Kearney et al., 2009;Suggitt et al., 2018).

ACK N OWLED G M ENTS
We

CO N FLI C T O F I NTER E S T S TATEM ENT
None declared.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used in the analysis of this paper can be found in the Dryad